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The primary afferent projection of the greater petrosal nerve to the solitary complex in the rat, revealed by transganglionic transport of horseradish peroxidase
Neuroscience Letters, 44 (1984) 13- 17 Elsevier Scientific Publishers Ireland Ltd.
13
NSL 02538
T H E P R I M A R Y A F F E R E N T P R O J E C T I O N OF THE G R E A T E R P E T R O S A L N E R V E TO THE SOLITARY C O M P L E X IN T H E RAT, R E V E A L E D BY T R A N S G A N G L I O N I C T R A N S P O R T OF H O R S E R A D I S H P E R O X I D A S E
YASUHIKO HOSOYA and YASUO SUGIURA Department of Anatomy, Institute of Basic Medical Sciences, University of Tsukuba, Niihari, lbaraki 305 (Japan)
(Received October 7th, 1983; Revised version received and accepted November 1st, 1983)
Key words. greater petrosal nerve - solitary complex - horseradish peroxidase
The primary afferent projection of the greater petrosal nerve (GPN) to the solitary complex was studied following application of horseradish peroxidase (HRP) to the GPN just distal to the geniculate ganglion. Labeled fibers were traced to the most rostral part of the solitary tract. Numerous collaterals entered the solitary complex from its dorsal and lateral aspects, and formed a dense plexus. They terminated in the dorsal half of the medial solitary nucleus at the level of the rostral half of the solitary complex, and in the ventrolateral and commissural nuclei at the level of the caudal half. The densest termination was observed in the medial solitary nucleus. Labeled terminals were found to contain round, clear synaptic vesicles and to make asymmetrical synaptic contacts with dendritic profiles. Studies using a silver i m p r e g n a t i o n a n d a n a u t o r a d i o g r a p h i c tracing m e t h o d showed that afferent fibers o f the facial nerve t e r m i n a t e m a i n l y in the rostral half of the solitary complex in the rat [12, 13], cat [9, 12] a n d m o n k e y [3]. Despite the fact that the afferent c o m p o n e n t o f the facial nerve is c o m p o s e d o f two nerves, the c h o r d a t y m p a n i a n d the greater petrosal nerve (GPN), these studies did not distinguish between t e r m i n a t i o n sites o f these two afferent c o m p o n e n t s . Using the t r a n s g a n g l i o n i c horseradish peroxidase ( H R P ) m e t h o d , N o m u r a a n d M i z u n o [11] d e m o n s t r a t e d projections of the chorda t y m p a n i to the v e n t r o l a t e r a l a n d dorsomedial parts o f the solitary complex in the cat. The present study, utilizing the t r a n s g a n g l i o n i c t r a n s p o r t o f H R P , examines the site a n d m o d e o f t e r m i n a t i o n o f the afferent c o m p o n e n t o f the G P N in the solitary complex in the rat. F o u r t e e n male Wistar rats (270-350 g in b o d y weight) were used for this study. The G P N was exposed b e h i n d the tensor t y m p a n i muscle in the t y m p a n i c cavity. A f t e r the nerve was t o r n with a fine needle along its course, a small piece of gelatinsponge soaked with 40°7o H R P (Sigma, type VI) was applied to the nerve several times, a n d finally the nerve a n d gelatin-sponge were sealed with super glue ( A r o n Alpha). A f t e r a survival time o f 2 days, the a n i m a l s were deeply anesthetized a n d perfused by R i n g e r ' s solution, followed by a fixative a n d 1070 sucrose in 0.1 M 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
14 phosphate buffer. The fixative was composed of 10% f o r m a l i n , 1°70 glutaraldehyde a n d 1% sucrose in 0.1 M p h o s p h a t e buffer. Transverse, h o r i z o n t a l or sagittal sections were cut serially t h r o u g h the lower b r a i n s t e m either at 100 ~m thick on a V i b r a t o m e or at 50 ~m thick o n a freezing m i c r o t o m e . These sections were processed for H R P histochemistry according to the m e t h o d of A d a m s [1]. Slices c o n t a i n i n g the solitary complex were t r i m m e d from the V i b r a t o m e sections after H R P histochemistry in 4 cases, postfixed with 2°70 OsO4, and processed for electron microscopy. The solitary complex was divided into several subnuclei according to the description given by Loewy a n d B u r t o n [10]. H R P - l a b e l e d afferent fibers were observed ipsilaterally along the ventral surface of the V I I l t h nerve. R u n n i n g dorsally o n the lateral surface of the spinal tract of the Vth nerve, labeled fibers were divided into several small fasciculi. They traversed
Fig. 1. Charts showing the labeled GPN afferents in the frontal plane. Sections A-K are arranged rostrocaudally at about 400 tzm intervals. HRP-labeled fibers and terminals are represented by dashed lines and black dots, respectively. AP, area postrema; CI and Cm, lateral and medial cuneate nucleus; DMV, dorsal motor nucleus of the vagus nerve; Fg, genu of the facial nerve; FLM, fasciculus longitudinalis medialis; Gn, gracile nucleus; Hy, hypoglossal nucleus; ICP, inferior cerebellar peduncle: Sn and St, solitary complex and tract; T, spinal tract of the trigeminal nerve; Vi, V1 and Vm, inferior, lateral and medial vestibular nucleus.
15
the dorsal half of the spinal tract of the Vth nerve and collected again into a compact fiber bundle at the area just dorsomedial to the spinal tract and nucleus of the Vth nerve (Fig. 1A, B). A small contingent of the labeled fibers ran further medially and terminated in the most dorsal region of the nucleus reticularis parvocellularis just lateral to the ascending root of the VIIth nerve, in which a part of the dorsal population of the efferent neurons of the GPN was located [4, 7]. The majority of the labeled fibers descended the border region between the solitary complex and the inferior vestibular nucleus, where the most dorsal part of the solitary tract appeared
Fig. 2. a: a photomicrograph showing labeled afferent fibers and their termination in the solitary complex, depicted from the same level as D of Fig. 1. Left side is medial and top is dorsal. Afferent fiber bundles run rostrocaudally in the solitary tract (arrow-heads) and give off m a n y fine collaterals (arrows) to the dorsal half of the medial solitary nucleus (asterisks). b: photomicrograph of a sagittal section of the solitary complex showing the labeled afferent terminals (arrows). c: an electron micrograph of a labeled myelinated fiber (arrow) and unmyelinated fibers (arrow-heads) in the solitary complex, d: an electron micrograph of a labeled terminal containing round synaptic vesicles and making an asymmetrical synaptic contact with a dendritic profile.
16 to be located (Fig. 2a, arrow-heads). The labeled descending fibers in the solitary tract gave off fine collaterals at right angles (Fig. 2a, arrows). After running for a short distance without branching, each collateral entered the solitary complex from dorsal, lateral and ventrolateral aspects. Within the solitary complex they ran tortuously, branched several times and terminated in varicosities of terminal boutons, about 1 t~m in diameter (Fig. 2b, arrows). At the level of the rostral half of the solitary complex, termination was sparse in the ventral half while it was heaviest in the dorsal half of the medial solitary nucleus (Figs. 1C-E and 2a, asterisks), which appeared to correspond to the dorsal 'gelatinous' part of Kalia and Sullivan [8]. Bundles of unlabeled coarse fibers located in the medial solitary nucleus were encircled by a dense network of the labeled collaterals and terminal fibers. In more caudal sections, terminal-labelings decreased in number and disappeared from the medial solitary nucleus at the level just rostral to the area postrema, while a moderate amount of termination could be observed in the ventrolateral nucleus (Fig. 1G-I). At the level just caudal to the area postrema, a few labeled terminals were observed ipsilaterally in the most dorsolateral region of the commissural nucleus (Fig. I J, K). No termination was observed in the lateral solitary nucleus, the parvocellular solitary nucleus and the dorsal motor nucleus of the vagus nerve. At the electron microscopic level, HRP-labeled fibers and terminals could easily be identified by their darker appearances and other ultrastructural features similar to those reported previously [2, 5, 6]. In the neuropil there were labeled small myelinated fibers (about 1 ~m in diameter) with fusiform or distorted oval contours (Fig. 2c, arrow) and labeled unmyelinated fibers (Fig. 2c, arrow-heads), about onethird of the size of the labeled myelinated fibers. The labeled unmyelinated fibers could occasionally be followed to labeled terminals. The labeled terminals contained round and clear synaptic vesicles, a small number of cored vesicles and one or two mitochondria (Fig. 2d). They made synaptic contacts with dendritic shafts or spinelike protrusions, associated with asymmetrical membrane thickenings. The present study demonstrates that the afferent fibers of the G P N , which are supposed to convey taste impulses from the palate, terminate in the dorsal half of the medial solitary nucleus, the ventrolateral solitary nucleus and the commissural nucleus. Except for the commissural nucleus, the termination areas seem to overlap with those of the chorda tympani reported for the cat [11]. Furthermore, the study shows that the terminals of G P N afferents make asymmetrical synaptic contacts with dendrites of solitary nucleus neurons. We are grateful to Dr. M. Matsushita for his valuable suggestions in preparing the manuscript. This study was supported by a grant from the University of Tsukuba. 1 Adams, J.C., Technical considerations on the use of horseradish peroxidase as a neuronal marker, Neuroscience, 2 (1977) 141-145.
17 2 Beattie, M.S., Bresnahan, J.C. and King, J.S., Ultrastructural identification of dorsal root primary afferent terminals after anterograde filling with horseradish peroxidase, Brain Res., 153 (1978) 127-134. 3 Beckstead, R.M. and Norgren, R., An autoradiographic examination of the central distribution of the trigeminal, facial, glossopharyngeal, and vagal nerves in the monkey, J. comp. Neurol., 184 (1979) 455-472. 4 Contreras, R.J., Gomez, M.M. and Norgren, R., Central origins of cranial nerve parasympathetic neurons in the rat, J. comp. Neurol., 190 (1980) 373-394. 5 Gwyn, D.G., Wilkinson, P.H. and Leslie, R.A., The ultrastructural identification of vagal terminals in the solitary nucleus of the cat after anterograde labelling with horseradish peroxidase, Neurosci. Lett., 28 (1982) 139-143. 6 Holstege, J.C. and Dekker, J.J., Electron microscopic identification of mammillary body terminals in the rat's AV thalamic nucleus by means of anterograde transport of HRP. A quantitative comparison with the EM degeneration and EM autoradiographic technique, Neurosci. Lett., 11 (1979) 129-135. 7 Hosoya, Y., Matsushita, M. and Sugiura, Y., A direct hypothalamic projection to the superior salivatory nucleus neurons in the rat. A study using anterograde autoradiographic and retrograde HRP methods, Brain Res., 266 (1983) 329-333. 8 Kalia, M. and Sullivan, J.M., Brainstem projections of sensory and motor components of the vagus nerve in the rat, J. comp. Neurol., 211 (1982) 248-264. 9 Kerr, F.W.L., Facial, vagal and glossopharyngeal nerves in the cat, Arch. Neurol., 6 (1962) 264-281. I0 Loewy, A.D. and Burton, H., Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat, J. comp. Neurol., 181 (1978) 421-450. 11 Nomura, S. and Mizuno, N., Central distribution of afferent and efferent components of the chorda tympani in the cat as revealed by the horseradish peroxidase method, Brain Res., 214 (1981) 229-237. 12 Rhoton, A.L., Jr., Afferent connections of the facial nerve, J. comp. Neurol., 133 (1968) 89-100. 13 Torvik, A., Afferent connections to the sensory trigeminal nuclei, the nucleus of the solitary tract and adjacent structures, J. comp. Neurol., 106 (1956) 51-132.
13
NSL 02538
T H E P R I M A R Y A F F E R E N T P R O J E C T I O N OF THE G R E A T E R P E T R O S A L N E R V E TO THE SOLITARY C O M P L E X IN T H E RAT, R E V E A L E D BY T R A N S G A N G L I O N I C T R A N S P O R T OF H O R S E R A D I S H P E R O X I D A S E
YASUHIKO HOSOYA and YASUO SUGIURA Department of Anatomy, Institute of Basic Medical Sciences, University of Tsukuba, Niihari, lbaraki 305 (Japan)
(Received October 7th, 1983; Revised version received and accepted November 1st, 1983)
Key words. greater petrosal nerve - solitary complex - horseradish peroxidase
The primary afferent projection of the greater petrosal nerve (GPN) to the solitary complex was studied following application of horseradish peroxidase (HRP) to the GPN just distal to the geniculate ganglion. Labeled fibers were traced to the most rostral part of the solitary tract. Numerous collaterals entered the solitary complex from its dorsal and lateral aspects, and formed a dense plexus. They terminated in the dorsal half of the medial solitary nucleus at the level of the rostral half of the solitary complex, and in the ventrolateral and commissural nuclei at the level of the caudal half. The densest termination was observed in the medial solitary nucleus. Labeled terminals were found to contain round, clear synaptic vesicles and to make asymmetrical synaptic contacts with dendritic profiles. Studies using a silver i m p r e g n a t i o n a n d a n a u t o r a d i o g r a p h i c tracing m e t h o d showed that afferent fibers o f the facial nerve t e r m i n a t e m a i n l y in the rostral half of the solitary complex in the rat [12, 13], cat [9, 12] a n d m o n k e y [3]. Despite the fact that the afferent c o m p o n e n t o f the facial nerve is c o m p o s e d o f two nerves, the c h o r d a t y m p a n i a n d the greater petrosal nerve (GPN), these studies did not distinguish between t e r m i n a t i o n sites o f these two afferent c o m p o n e n t s . Using the t r a n s g a n g l i o n i c horseradish peroxidase ( H R P ) m e t h o d , N o m u r a a n d M i z u n o [11] d e m o n s t r a t e d projections of the chorda t y m p a n i to the v e n t r o l a t e r a l a n d dorsomedial parts o f the solitary complex in the cat. The present study, utilizing the t r a n s g a n g l i o n i c t r a n s p o r t o f H R P , examines the site a n d m o d e o f t e r m i n a t i o n o f the afferent c o m p o n e n t o f the G P N in the solitary complex in the rat. F o u r t e e n male Wistar rats (270-350 g in b o d y weight) were used for this study. The G P N was exposed b e h i n d the tensor t y m p a n i muscle in the t y m p a n i c cavity. A f t e r the nerve was t o r n with a fine needle along its course, a small piece of gelatinsponge soaked with 40°7o H R P (Sigma, type VI) was applied to the nerve several times, a n d finally the nerve a n d gelatin-sponge were sealed with super glue ( A r o n Alpha). A f t e r a survival time o f 2 days, the a n i m a l s were deeply anesthetized a n d perfused by R i n g e r ' s solution, followed by a fixative a n d 1070 sucrose in 0.1 M 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
14 phosphate buffer. The fixative was composed of 10% f o r m a l i n , 1°70 glutaraldehyde a n d 1% sucrose in 0.1 M p h o s p h a t e buffer. Transverse, h o r i z o n t a l or sagittal sections were cut serially t h r o u g h the lower b r a i n s t e m either at 100 ~m thick on a V i b r a t o m e or at 50 ~m thick o n a freezing m i c r o t o m e . These sections were processed for H R P histochemistry according to the m e t h o d of A d a m s [1]. Slices c o n t a i n i n g the solitary complex were t r i m m e d from the V i b r a t o m e sections after H R P histochemistry in 4 cases, postfixed with 2°70 OsO4, and processed for electron microscopy. The solitary complex was divided into several subnuclei according to the description given by Loewy a n d B u r t o n [10]. H R P - l a b e l e d afferent fibers were observed ipsilaterally along the ventral surface of the V I I l t h nerve. R u n n i n g dorsally o n the lateral surface of the spinal tract of the Vth nerve, labeled fibers were divided into several small fasciculi. They traversed
Fig. 1. Charts showing the labeled GPN afferents in the frontal plane. Sections A-K are arranged rostrocaudally at about 400 tzm intervals. HRP-labeled fibers and terminals are represented by dashed lines and black dots, respectively. AP, area postrema; CI and Cm, lateral and medial cuneate nucleus; DMV, dorsal motor nucleus of the vagus nerve; Fg, genu of the facial nerve; FLM, fasciculus longitudinalis medialis; Gn, gracile nucleus; Hy, hypoglossal nucleus; ICP, inferior cerebellar peduncle: Sn and St, solitary complex and tract; T, spinal tract of the trigeminal nerve; Vi, V1 and Vm, inferior, lateral and medial vestibular nucleus.
15
the dorsal half of the spinal tract of the Vth nerve and collected again into a compact fiber bundle at the area just dorsomedial to the spinal tract and nucleus of the Vth nerve (Fig. 1A, B). A small contingent of the labeled fibers ran further medially and terminated in the most dorsal region of the nucleus reticularis parvocellularis just lateral to the ascending root of the VIIth nerve, in which a part of the dorsal population of the efferent neurons of the GPN was located [4, 7]. The majority of the labeled fibers descended the border region between the solitary complex and the inferior vestibular nucleus, where the most dorsal part of the solitary tract appeared
Fig. 2. a: a photomicrograph showing labeled afferent fibers and their termination in the solitary complex, depicted from the same level as D of Fig. 1. Left side is medial and top is dorsal. Afferent fiber bundles run rostrocaudally in the solitary tract (arrow-heads) and give off m a n y fine collaterals (arrows) to the dorsal half of the medial solitary nucleus (asterisks). b: photomicrograph of a sagittal section of the solitary complex showing the labeled afferent terminals (arrows). c: an electron micrograph of a labeled myelinated fiber (arrow) and unmyelinated fibers (arrow-heads) in the solitary complex, d: an electron micrograph of a labeled terminal containing round synaptic vesicles and making an asymmetrical synaptic contact with a dendritic profile.
16 to be located (Fig. 2a, arrow-heads). The labeled descending fibers in the solitary tract gave off fine collaterals at right angles (Fig. 2a, arrows). After running for a short distance without branching, each collateral entered the solitary complex from dorsal, lateral and ventrolateral aspects. Within the solitary complex they ran tortuously, branched several times and terminated in varicosities of terminal boutons, about 1 t~m in diameter (Fig. 2b, arrows). At the level of the rostral half of the solitary complex, termination was sparse in the ventral half while it was heaviest in the dorsal half of the medial solitary nucleus (Figs. 1C-E and 2a, asterisks), which appeared to correspond to the dorsal 'gelatinous' part of Kalia and Sullivan [8]. Bundles of unlabeled coarse fibers located in the medial solitary nucleus were encircled by a dense network of the labeled collaterals and terminal fibers. In more caudal sections, terminal-labelings decreased in number and disappeared from the medial solitary nucleus at the level just rostral to the area postrema, while a moderate amount of termination could be observed in the ventrolateral nucleus (Fig. 1G-I). At the level just caudal to the area postrema, a few labeled terminals were observed ipsilaterally in the most dorsolateral region of the commissural nucleus (Fig. I J, K). No termination was observed in the lateral solitary nucleus, the parvocellular solitary nucleus and the dorsal motor nucleus of the vagus nerve. At the electron microscopic level, HRP-labeled fibers and terminals could easily be identified by their darker appearances and other ultrastructural features similar to those reported previously [2, 5, 6]. In the neuropil there were labeled small myelinated fibers (about 1 ~m in diameter) with fusiform or distorted oval contours (Fig. 2c, arrow) and labeled unmyelinated fibers (Fig. 2c, arrow-heads), about onethird of the size of the labeled myelinated fibers. The labeled unmyelinated fibers could occasionally be followed to labeled terminals. The labeled terminals contained round and clear synaptic vesicles, a small number of cored vesicles and one or two mitochondria (Fig. 2d). They made synaptic contacts with dendritic shafts or spinelike protrusions, associated with asymmetrical membrane thickenings. The present study demonstrates that the afferent fibers of the G P N , which are supposed to convey taste impulses from the palate, terminate in the dorsal half of the medial solitary nucleus, the ventrolateral solitary nucleus and the commissural nucleus. Except for the commissural nucleus, the termination areas seem to overlap with those of the chorda tympani reported for the cat [11]. Furthermore, the study shows that the terminals of G P N afferents make asymmetrical synaptic contacts with dendrites of solitary nucleus neurons. We are grateful to Dr. M. Matsushita for his valuable suggestions in preparing the manuscript. This study was supported by a grant from the University of Tsukuba. 1 Adams, J.C., Technical considerations on the use of horseradish peroxidase as a neuronal marker, Neuroscience, 2 (1977) 141-145.
17 2 Beattie, M.S., Bresnahan, J.C. and King, J.S., Ultrastructural identification of dorsal root primary afferent terminals after anterograde filling with horseradish peroxidase, Brain Res., 153 (1978) 127-134. 3 Beckstead, R.M. and Norgren, R., An autoradiographic examination of the central distribution of the trigeminal, facial, glossopharyngeal, and vagal nerves in the monkey, J. comp. Neurol., 184 (1979) 455-472. 4 Contreras, R.J., Gomez, M.M. and Norgren, R., Central origins of cranial nerve parasympathetic neurons in the rat, J. comp. Neurol., 190 (1980) 373-394. 5 Gwyn, D.G., Wilkinson, P.H. and Leslie, R.A., The ultrastructural identification of vagal terminals in the solitary nucleus of the cat after anterograde labelling with horseradish peroxidase, Neurosci. Lett., 28 (1982) 139-143. 6 Holstege, J.C. and Dekker, J.J., Electron microscopic identification of mammillary body terminals in the rat's AV thalamic nucleus by means of anterograde transport of HRP. A quantitative comparison with the EM degeneration and EM autoradiographic technique, Neurosci. Lett., 11 (1979) 129-135. 7 Hosoya, Y., Matsushita, M. and Sugiura, Y., A direct hypothalamic projection to the superior salivatory nucleus neurons in the rat. A study using anterograde autoradiographic and retrograde HRP methods, Brain Res., 266 (1983) 329-333. 8 Kalia, M. and Sullivan, J.M., Brainstem projections of sensory and motor components of the vagus nerve in the rat, J. comp. Neurol., 211 (1982) 248-264. 9 Kerr, F.W.L., Facial, vagal and glossopharyngeal nerves in the cat, Arch. Neurol., 6 (1962) 264-281. I0 Loewy, A.D. and Burton, H., Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat, J. comp. Neurol., 181 (1978) 421-450. 11 Nomura, S. and Mizuno, N., Central distribution of afferent and efferent components of the chorda tympani in the cat as revealed by the horseradish peroxidase method, Brain Res., 214 (1981) 229-237. 12 Rhoton, A.L., Jr., Afferent connections of the facial nerve, J. comp. Neurol., 133 (1968) 89-100. 13 Torvik, A., Afferent connections to the sensory trigeminal nuclei, the nucleus of the solitary tract and adjacent structures, J. comp. Neurol., 106 (1956) 51-132.